Systems, methods, and devices for frozen sample distribution

ABSTRACT

A drilling system including a motor that produces a sonic, linear oscillatory motion is provided for removing a frozen biological sample from a stored frozen specimen and methods of use thereof without thawing the remainder of the specimen. The stator and slider assembly is operated by a servo controller which can communicate and be programmed through a port of a PC equipped with software.

RELATED APPLICATIONS

The present application is related to and claims the benefit of U.S.application Ser. No. 13/901,045 filed May 23, 2013, now U.S. Pat. No.8,713,948 issue date May 6, 2014, which is a continuation of U.S.application Ser. No. 12/087,695, issue date May 28, 2013 as U.S. Pat.No. 8,448,456, which was a national phase of PCT application havingserial number PCT/US2007/001094, filed in the PCT Receiving Office ofthe U.S. Patent and Trademark Office Jan. 16, 2007, claiming priority ofU.S. provisional patent application Ser. No. 60/758,807 filed Jan. 13,2006, each of which is hereby incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to a coring system for extracting frozenbiological samples from frozen specimens without thawing the specimen,and methods of use thereof.

GOVERNMENT SUPPORT

This invention was made with government support under CAI 14167 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

Stored biological samples in biorepositories are an important resourcefor research into the diagnosis and etiology of diseases. Biologicalspecimens are stored in military facilities, forensic DNA banks,government laboratories, diagnostic pathology and cytology laboratories,commercial enterprises, and nonprofit organizations such as hospitals.Preservation and analysis of biological samples increases knowledgeabout diseases and provides a basis for developing better processes toprevent, diagnose, and treat these diseases.

A conservative estimate shows that 282 million specimens are currentlystored in U.S. facilities, and the rate has been increasing at 20million specimens per year beginning since 1998 (National BioethicsAdvisory Commission, “Research Involving Human Biological Materials:Ethical Issues and Policy Guidance”, Volume 1 Report and Recommendationsof the National Bioethics Advisory Commission, Rockville, Md., August1999). Specimens are stored as slides, paraffin blocks, formalin-fixed,tissue cultures, or extracted DNA. Research in fields such as cancer,infectious diseases, and mental disorders is advanced by quick access tosuch materials.

Biological samples are typically stored frozen, for example at about−20° C., −40° C., or at about −80° C. in cryotubes. Current industrymethods of aliquotting samples involve thawing and refreezing thestorage volume of the specimen each time an aliquot is removed.Laboratory results indicate that repeated freeze-thaw cycles degrade thespecimen. However, freezing (cryostorage) extends the usable life ofbiological samples.

Further, current methods for accessing and processing samples involvedelay and are a bottleneck in the use of these repositories. The sampleis exposed to repeated freeze/thaw cycles that degrade the sample.Alternatively, storage of one specimen divided into multiple containersprior to freezing results in the inefficient use of freezer space andpotential increased costs of cryotubes, labels, and time, and introducesa new source of error in processing.

An efficient method for obtaining samples (aliquots) from frozenbiological specimen from storage volumes, i.e., reducing the need forexcess freezer storage space, reducing lead-times for receivingspecimens, and reducing degradation of specimens is needed.

SUMMARY

The invention in certain aspects provides an apparatus for obtaining afrozen sample from a frozen biological specimen, the apparatus includes:a drill for contacting the specimen within a container, wherein thedrill includes a motor that actuates a hollow bit coring needle; and agrip for holding the specimen, in which the grip positions an open endof the container proximal to the needle, wherein the motor impels theneedle into the specimen within the container, in which the frozensample is removed from the specimen absent thawing of the specimenretained in the container.

In certain embodiments, the apparatus further includes a coolingreservoir to immerse a closed end of the container. It is envisionedthat the cooling reservoir keeps the specimen frozen to avoiddegradation; keeps the deposited sample frozen; prevents build-up offrost on the surface of the specimen; prevents addition of water to thespecimen; and allows the operator to see the sample.

In a related embodiment of the apparatus the container is at least oneselected from a cryotube, an array, a compartment in a molded recipientblock, or a platform.

In certain embodiments of the apparatus, the motor provides a linearoscillatory motion. In alternative embodiments of the apparatus, themotor provides a rotary motion.

In another embodiment, the apparatus further includes a servo controllerthat communicates with a computer and is programmable with the computerand software. In an alternative embodiment, the apparatus is manuallycontrolled.

In certain embodiments of the apparatus, the motor provides a force forthe bit for striking the frozen specimen of about 30 to about 90 N, ofabout 35 to about 80 N, or of about 40 to about 75 N or the like, andcompressive stresses of about 40 MPa to about 80 MPa, or of about 50 MPato about 70 MPa or the like, and wherein the specimen is maintained at atemperature of about −90° C., or about −80° C., or about −70° C., orabout −40° C. or about −20° C. or about −10° C., i.e., is a range ofabout −90° C. to about −10° C. or the like. Due to the cooling elementsof the apparatus herein the buildup of frost on the specimen does notoccur.

In a certain embodiment, the specimen has a volume of about 0.5 mL toabout 5 mL, or of about 1.5 mL to about 15 mL, or of about 5 mL to about50 mL or the like. In another embodiment, the sample has a volume ofabout 10 μl to about 50 μl or about 54 μl to about 100 μl or about 100μl to about 500 μl or about 500 μl to about 1.0 mL.

In another embodiment of the apparatus, the drill bit needle furtherincludes a distal tip having a ground point and lifting teeth. Incertain embodiments of the apparatus the drill bit needle is ceramic ormetal, for example, prefabricated standard surgical tubing including atleast one metal selected from the group of, titanium, INCONEL 625,stainless steel 304, stainless steel 304L, stainless steel 316, andstainless steel 316L.

In another embodiment, the apparatus further includes a receiving devicefor the sample. In a related embodiment, the receiving device is amicroarray, for example, the microarray is a recipient block havingpre-molded cavities. The pre-molded cavities have a taper that enlargesthe cavity towards the bottom of the cavity, for example, a taper toprevent the samples from falling out. In certain embodiments, thepre-molded cavities have a 2° taper or about a 5° taper, or about a 10°taper

In certain embodiments, the apparatus further includes an ejection pinwith a spring return or other type of automatic return, in which theejection pin has a cross-sectional diameter less than and substantiallysimilar to the inner diameter of the needle.

In another embodiment, the apparatus further includes a housing for atleast one selected from the group of humidity control, refrigeration,operator safety from bloodborne pathogens, and sterility.

Another aspect of the invention herein is a method for obtaining afrozen sample from a frozen biological specimen and maintaining thespecimen in a frozen condition, the method involving: contacting thespecimen within a container with a drill having a motor that actuates ahollow bit coring needle; and a grip for holding the specimen, whereinthe grip positions an open end of the container proximal to the needle,wherein actuating the motor impels the needle into the specimen;retracting the needle containing the frozen sample; and impelling thefrozen sample from the needle to a receiving device, in which the frozensample has been obtained from the frozen biological specimen and thespecimen has been maintained in a frozen condition.

In another embodiment, the method further includes a cooling reservoirfor immersing a closed end of the container. In a related embodiment thecontainer is at least one selected from a cryotube, a compartment in anarray, or a platform.

In certain embodiments, the method is performed in a local environmentat a temperature of at least about −90° C. to about −10° C. or othertemperatures that maintain the specimen in a frozen state. In anotherembodiment, the method further includes re-iterating the steps to obtaina duplicate aliquot of the sample. In a related embodiment, the methodfurther includes re-iterating the steps to obtain a sample from at leastone additional specimen.

In certain embodiments of the method, the motor that actuates a hollowbit drilling needle imparts a linear oscillatory motion to fracture thesurface of the specimen.

In another embodiment of the method, impelling the frozen sample fromthe needle to a receiving device comprises actuating an ejection pin.

In another embodiment of the method, the receiving device is selectedfrom the group consisting of an assay tube; a cryotube; a planar surfacefor pathology, histology and/or micro-array analysis; and a well of amulti-well dish.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing a drill bit (TechnoDiamant) with aninside diameter (ID) of 0.0395 mm and an outside diameter (OD) of 0.070mm. The tip includes a diamond/metal matrix.

FIG. 2 is a plot of temperature of the sample in the hollow drill bit asa function of time. The method involved pre-cooling the bit using thecoolant in the reservoir, establishing thermal equilibrium, andexposuring to air. The data are the average of four experiments.

FIG. 3 is a diagram of a linear oscillating motor with drill bit,showing the driving system (31), slider (32), and bit (33).

FIG. 4 is a photograph showing a frozen cell line array made using amold for frozen tissue arraying of samples obtained by the methodsherein. FIG. 4 panel A shows the cell line array. The prototype blockincludes 12 cavities. The tissue microarray contains 8 samples, 5 oftissue (light color) which are in the first two rows and 3 dyed OCT(Optimal Cutting Temperature) samples (dark color), which are in thethird row. There were four empty cavities to show the shape of thecavity. Some of the samples are slightly darker because of thelightening. This is because they were ejected deeper into the moldedholes. The molded holes have a 2° taper to prevent the samples fromfalling out. FIG. 4 panel B shows a stained sample. FIG. 4 panel C is aphotomicrograph of cells from a sample stained with an antibody specificfor the phosphorylated form of ERK.

FIG. 5 is a photograph of frozen tissue microarray (TMA) stained withFITC-labeled Hepsin peptide. FIG. 5 panel A is a photomicrograph ofcells from a sample of frozen tumor glands (20×) showing specificmembranous staining near the lumen of the cancer glands. FIG. 5 panel Bis a photomicrograph of a control frozen large benign gland (20×) thatis negative for specific staining showing weak non-specific staining inthe stroma.

FIG. 6 is a cross section of an end of a coring bit needle having groundpoints and lifting teeth and an ejector pin for ejecting a frozen samplefrom the needle.

FIG. 7 is a schematic illustration of one embodiment of a system havinga grip in the form of a block having premolded cavities for holding acontainer having a frozen specimen therein in a cooling reservoir whilea servo motor produces linear oscillatory motion of the coring bitneedle.

FIG. 8 is a schematic illustration of one embodiment of a system havinga grip for holding a container having a frozen specimen therein in acooling reservoir while a servo motor produces rotary motion of thecoring bit needle.

DETAILED DESCRIPTION

The preservation and analysis of repositories of frozen biologicalspecimens increases knowledge about diseases and provides a basis fordeveloping better processes to prevent, diagnose, and treat thesediseases.

Methods and apparatus provided herein use subsonic impact drilling intothe still frozen specimens to extract sample (aliquots). The apparatusreduces the time required to obtain the samples by automating a processof extracting aliquot frozen samples, and requires less training andoperation than current industry procedure. The apparatus and methodsherein reduce lead time for laboratory researchers, speed medicalresearch, and most important, provide for biological stability andintegrity of the stored frozen specimens.

Industry Sample Processing

Biological repositories contain many frozen biological tissue and fluidspecimens that are available to scientists to support research onbiomarkers, nutrition, functional genomics, as well as other researchareas. For example, blood banks collect approximately 15 million unitsof blood a year with 20,000 to 40,000 units stored for future use.Current biorepository facilities operate under prohibitively long leadtimes for delivery of ordered sample sets. For example, in the Nurses'Health Study (NHS) aliquotting operation, a research assistant processed150 samples a week and a typical study consists of approximately 1000samples. Therefore, it takes a research assistant more than six weeks inaddition to another 1-2 weeks of data management to complete sampleprocessing for a single study.

It is known that cycles of freeze-thawing result in the degradation ofcritical biological molecules such as RNA and proteins in the samples.This is troubling for researchers because the exact extent ofdegradation of the sample has not been quantified. Cryostorage isnecessary to extend the usable life of biological specimens. To ensurethat the highest quality samples reach the laboratories, repeated cyclesof freeze-thawing must be minimized or eliminated.

One approach is characterized by storing samples in one container andaliquotting the required amount when requested. The sample is stored ina large cryotubes for example, a 1.8 mL or 4.5 mL cryotubes, and then isfrozen. When a sample is requested the entire sample must be thawed,mixed, and requested aliquots are then removed. The remaining sample isrefrozen in the original container. This method is an efficient use oflabor, space, and energy because the sample is stored in one container.The drawback is the sample is exposed to many freeze/thaw cycles whichdegrade the sample and reduces its future value.

Another method of storage and sampling is storing the sample in aliquotsized containers. This method has the benefit of limited degradation,however it is an inefficient use of labor, freezer space and energybecause many cryotubes need to be stored per sample. Further, there isadded potential for confusion of identification of samples.

The third process of storing samples is a combination of the twopreviously presented methods. This hybrid method begins with mass frozenstorage in, for example 1.8 mL or 4.5 mL cryotubes. After a request foranalysis, a specimen is separated into, for example aliquots, and unusedaliquots are then refrozen. This storage method of the specimensexperience only two rounds of freeze-thawing. The utilization of freezerspace is minimized until the specimen has been requested. This approachhowever compromises freezer space versus specimen quality, and may leadto confusion, and is labor intensive.

Introducing a frozen specimen aliquotting instrument to biologicalrepositories will moderate the need for excess freezer storage space,reduce lead times for receiving specimen aliquots, minimize trainingneeds, and limit degradation of specimens through reduction of repeatedcycles of freeze-thawing. The proposed instrument directly samples thefrozen specimen without the need for thawing. The required freezer spacewill be minimized because the specimen can be stored in larger volumes.The method of sampling or aliquotting through the automation of drillingfrozen specimens introduces a significant improvement to the currentsampling operations.

As used herein, the term “specimen” refer to stored frozen biologicalsolid tissues and bodily fluids. The term “sample” and “aliquot” referto portions of an initial specimen.

Design Objectives

A design is needed that will extract a one millimeter diameter core froma frozen biological specimen that will be maintained at or below about−90° C., −80° C., or −70° C., −40° C., or −20° C. Examples show that afrozen specimen exhibits the behavior of a brittle material. The mannerin which the specimens are expected to fail and the various modes ofheat generation are analyzed herein. Rotary coring methods were found inone preliminary test to be unsatisfactory because of high buckling andtorsional stresses in the hit transmitted to the biological specimen.

An ultrasonic drilling system including piezoelectric stacks wascompared to a subsonic system driven by a linear motor. The amount ofheat generation was found to be directly proportional to frequency andstroke length, and the ultrasonic method was discounted as a potentialsolution through calculations and analysis.

A linear motor was then analyzed. An initial model tested was thatdesigned by the LinMot Corporation and was chosen to provide the motionrequired of the design concept. Testing was performed with various bitgeometries and an optimized, drill bit design was found. A sinusoidal,sonic motion at a frequency of 50 Hz and a 2 mm stroke was determined toprovide the force necessary to initiate fracture within the sample. FIG.3 shows a LinMot Linear motor with drilling bit.

The objective for the apparatus and methods provided herein is to obtaina sample from a frozen biological specimen. This objective presents asignificant challenge resulting from the frozen specimen's fracturemechanics and the system's temperature constraints. Data from examplesshow that frozen saline is representative of the actual biologicalspecimens and exhibit brittle characteristics. Through investigation, itwas found that the brittle fracture mode results in formation of icechips as a sample is then extracted. To assist in ice chip clearance,and allow for a deeper core an oscillatory motion was considered.Analysis of this type of apparatus determined that heat generation fromfriction was proportional to frequency and stroke length. Thereforeobjectives of further examples were to determine an oscillatory motionhaving low frequency and stroke, providing the force necessary to causefracture within the sample. This motion is herein combined with a uniquebit design to meet these objectives.

Design Parameters

Described parameters for the device include: volumetric uniformitymaintained between aliquots; storage volumes utilized in an efficientmatter, minimizing wasted sample; and minimal time and user inputcompared to prior art methods.

Described aliquot volume requirements based on laboratory testingcriteria and industry efficiency requirements include at least about 10μL, 50 μL, 100 μL, or about 500 μL for a successful analysis. In somecases, industry standards limit the aliquot volume to less than about120 μL for efficient use of stored volumes. An exemplary cryotubeholding 1.8 mL yields about 10 aliquots to meet industry efficiencystandards. Peripheral damage to the surrounding specimen should beminimized to enable additional samples to be obtained from the samecryotube.

Sampling Methods

Rotary drilling using rotary motion to drive a drill bit into a materialis shown in U.S. patent application number 2002/0129975 (Barth et al.).This ice auger system is in use for cutting and removing a solid corefrom frozen water, such as lakes and rivers. The purpose is to create abore in the ice to remove a cylindrical ice core without significantlycracking the ice while reducing ice shavings. The system rotates by theuse of a motor device connected to the upper end of the elongated shaft.The system cores with at least one bit for engaging the ice and usesfighting for continuous cutting. This system is not designed forprecision obtaining a particular precise volume, or for maintaining theintegrity of the inner core and surrounding ice. This system uses thebasic rotary motion to cut and remove a solid core.

Ultra-subsonic corer/driller apparatus is shown in U.S. Pat. No.6,689,087 (Pal et al.). This system produces a hammering action by useof ultra-subsonic and subsonic vibrations to produce effective drillingand coring. The ultra-subsonic driller and corer is used in planetaryand geological exploration, military, and medical operations and is notdesigned for precision obtaining a particular precise volume, or formaintaining the integrity of the inner core and surrounding ice. Thissystem uses the basic rotary motion to cut and remove a solid core.

Microarray technology suffers from disadvantages of smaller needles thatbuckle more easily due to the reduced diameter and provide lessrepresentation of a tissue specimen, e.g., a cancer biopsy. The samplesizes of about 3 mm to 4 mm in length, taken from a frozen donor blockat about −20° C. can be obtained.

For some embodiments, diamond crystals are bonded under ultrahightemperatures and pressures to cutting elements as shown in U.S. Pat. No.5,437,343 (Cooley et al.). The thermally stable polycrystalline diamondcutting element is put to use on a rotary drag bit for drillingsubterranean formations in a shearing motion. Boron nitride and siliconnitride are also super-hard materials and equally able to be bonded tocutting elements. The use of either sputter-coating diamonds or heatenameling super-hard materials onto the tips of the needle tipgeometries will increase cycles to failure and maintain low initialcontact stresses on the needle tip.

Bit Design

As used herein, the terms “bit”, “drill bit”, or “needle” refer to ahollow tube attached to a linear oscillatory motor for impacting andboring into a frozen biological specimen and removing a frozen sample.

The bit (needle) geometry is minimized to control radial cracking duringdrilling to allow for the maximum number of samples to be removed fromeach cryotube. Factors that effect radial crack propagation includeimpact velocity, impact force, bit surface area, and pitch of the firstlifting tooth.

Reduction of stress exerted at the bit tip is an aspect and stresses onthe bit are minimized to prevent bit failure from fatigue. Thismaximizes the number of cores possible per needle reducing operatingcosts and making the system more efficient.

To minimize binding of the needle as it penetrates the frozen sample, afrozen ice chip removal system is integrated into the design of theneedle. This system facilitates removal of unwanted compacted frozen icechips. Clearing the impact area of loose frozen ice chips reducesfriction between the impact surfaces of the needle and the frozensample.

Material selection for the needle is affected by factors including,strength, the ability to withstand temperature cycling between ambientand that of the frozen specimen, low cost, and ease ofmanufacturability.

Exemplary Model

A preliminary design for a frozen tissue microarrayer was based on ahollow drilling device (a diamond core drill bit from TechnoDiamant)that cuts by a combination of axial force and rotation. This combinationof cutting modes reduces the stress levels in the needle compared to theuse of axial force alone as is done in the existing commerciallyavailable equipment. The lower forces improve needle life and reduce theimpact of coring on the architecture of the tissue. The needle seen inFIG. 1 was to cut 20 samples during the creation of a small frozentissue array.

This drilling device is integrated into an x-y milling table, whichoffers computer control of x-y-z position and on/off control of thedrill bit with fixed, but selectable, rotational speed. The millingtable, or mini-mill, has 0.003175 mm step resolution with repeatabilitybetter than 0.0127 mm, which is more than adequate for this application.Samples were obtained with the Technodiamant needle and the mini-mill infrozen saline and tissue at −70° C.

Temperature Control

Frozen tissue specimens are held in tissue cassettes inserted in aholder and place in a coolant reservoir mounted on a milling machine.The coolant reservoir contains a dry ice-alcohol slurry to maintain thetissues in a frozen condition, and in certain embodiments includes anair coolant loop that provides a stream of cool air above the tissueblock. This air circulation keeps the top of the block from warming andprevents buildup of frost on the block. This assembly was found suitableto maintain the block temperature at −70° C. or even colder, for atleast eight hours.

The bit gains heat by friction as it moves within the frozen specimenand also by convection as it moves from the specimen to the recipientblock. The bit is pre-cooled so that heat from friction is dissipatedinto the surrounding specimen. When used iteratively, the bit ispre-cooled in coolant before each use for example, the bit is contactedwith a cold air stream above the specimen.

To determine thermal properties of the system, a thermocouple treatedwith cryothermal grease to provide thermal contact was inserted into thebit which was chilled by contact with a dry ice/alcohol slurry. The bitwas then moved out of the slurry and temperature recorded as shown inFIG. 2. The data show that the temperature of the sample was maintainedat −20° C. for 100 s, sufficient to position and deposit the frozensample in the recipient container.

Insertion of the Tissue Sample into the Recipient Block

The frozen sample is removed from the needle 33 with an ejection pin 39(FIG. 6) having a very close fit to the inside diameter of the drillingneedle to completely and cleanly remove the frozen sample from theneedle. The pin further includes a spring return, and can include aTeflon seal to maximize sample recovery from the needle prior toiteration of the process. A tight fit between the Teflon piece at thedistal end of the drilling needle and the inside diameter of the needleminimizes retention, and external material is removed using a cleaningstation and the following steps: the drilling needle is inserted into abed of brushes covered in a 10% bleach solution into which the coringneedle is inserted. Once inserted the needle is moved around thebristles in an orbital motion while the needle is rotating and theejection pin is moved up and down and the Teflon piece is partiallyexposed to allow its perimeter to be cleaned as well; in the next stagethe method is similar to the herein except that the bleach is replacedby saline solution; then the station is dried to remove any residualsaline solution; and a cooling station brings the needle to the desiredtemperature range.

As illustrated in FIGS. 6-8, the apparatus includes: a drill 33contacting the specimen 49 within a container 41, wherein the drillincludes a motor 55 that actuates a hollow bit coring needle 33; and agrip 43 for holding the specimen, in which the grip positions an openend of the container proximal to the needle, wherein the motor drivesthe needle into the specimen within the container, such that a frozensample is removed from the specimen while the sample and the specimenare maintained in a frozen condition. The drill bit needle 33 includes adistal tip having a ground point 37 and lifting teeth 35, as illustratedin FIG. 6. FIG. 7 illustrates a system having a motor 55A that produceslinear oscillating motion of the coring bit needle 33. FIG. 8illustrates a system having a motor 55B that produces a rotary motion ofthe coring bit needle 33. The motors 55A, 55B in FIGS. 7 and 8 include aservo controller that communicates with a computer and is programmablewith the computer and software, as illustrated. The apparatus in FIGS. 7and 8 further includes a cooling reservoir 45 to immerse a closed end ofthe container 41. The systems illustrated in FIGS. 7 and 8 also includea cooling reservoir 45 for immersing a closed end of the container 41.As illustrated, the cooling reservoir 45 includes a coolant 47.

A cleaning assay was used as follows: ten samples of frozen human tissuetaken as herein with the cleaning procedure and ten controls without theherein cleaning procedure were tested using the QuantiBlot® Human DNAQuantitation Kit (Applied Biosystems), a probe that is complementary toa primate-specific alpha satellite DNA sequence at the D17Z1 locus.

Tissue Cassette

The frozen tissue arrayer cassette for the frozen specimens machinedfrom aluminum, for example, in one piece or in multiple pieces such thatthe cassette functions during freezing, storage, and subsequent arrayconstruction. Further, the same set can be used for the donor frozenspecimen or for the recipient block.

Maintaining Tissue Morphology

In the process of obtaining samples from the frozen specimens anddepositing these samples into the recipient block, the prior art methodsapply unwanted forces to the cell and/or tissue. Drilling applies ashear force (rotation of the coring bit through the tissue) and acompression force (as the coring needle is pushed into the tissue).These coring forces have the potential to “twist” the tissue andcompress the tissue. During insertion of the cores into the recipientblock two forces are applied, one is a force from the ejector needlethat is uniformly applied across the tissue surface and the other is afriction force at the intersection of the core with the inside diameterof the coring needle and the inside diameter of the cavity in therecipient block. These deposition forces have the potential to compressthe tissue and cause relative motion between the perimeter and thecenter of the core.

An objective is to optimize the drilling and deposition forces to apoint where there is no detectable alteration of the morphology of thetissue. The design variables under our control that impact on theseforces include: the geometry of the drilling needle, the rotationalspeed during drilling, the rate at which the needle advances duringdrilling, the force applied and the rate at which the ejection pinpushes on the tissue, the difference between the outside diameter of thedrill and the inside diameter of the cavity in the recipient block, thesurface finish of the needle, and the accuracy with which the drillingneedle is placed over the cavity in the recipient block. The positioningaccuracy is under computer control, to preserve the biochemical fidelityof the tissue temperature maintained at −70° C.

Piezoelectric force and torque sensor (PCB Piezotronics, force: PCB1102-05A and torque: PCB 2308-02A) installed in the arrayer measure theapplied force and torque profiles (force and torque versus time) duringthe drilling process. The profiles are analyzed in conjunction with thepathology evaluation (see Examples herein) to identify the optimaldesign conditions. The metrics that are used to assess the merits of thevariables include: morphology of the samples as a function of singletissue sections are evaluated to assess the impact of the drillingprocess. To perform the evaluation samples are taken from many differenttissue types and sections are taken from both the donor tissues and thefrozen tissue arrays and paired up. Double-blinded evaluations areperformed by pathologists.

Sections from the donor tissues and the frozen arrays are cut andprocessed for the three key assays identified herein (IHC, FISH-DNA and-RNA) and a blinded evaluation are used. Immunohistochemistry(IHC)/Immunofluorescence (IF) are techniques used to assess the presenceof a specific protein or antigen in cells by use of a specific antibody,which binds to it, thereby allowing visualization and examination undera microscope. The advantage of IF over IHC is that many differentfluorescent chromagens are available and thus different antigens can bedifferentially labeled and visualized at the same time.

(Fluorescence) in-situ hybridization are techniques that allow specificDNA/RNA sequences (e.g. genes) to be detected by use of labeledcomplementary DNA/RNA probes in morphologically preserved chromosomes,cells or tissue sections. This provides microscopic topologicalinformation to gene activity at the DNA, or mRNA level. Likewise herein,the advantage of FISH over ISH is that many different fluorochromes areavailable and thus different DNA/RNA sequences can be differentiallylabeled and visualized at the same time.

The invention having been fully described, it is further shown by thefollowing examples and claims, which are illustrative and are not meantto be further limiting. Those skilled in the art will recognize or beable to ascertain using no more than routine experimentation, numerousequivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references, including issued patents and publishedpatent applications, cited throughout this application are herebyincorporated by reference.

EXAMPLES Example 1 Determination of Required Force

A general objective is minimization of heat generation occurring as aresult of this process. Experimental simulations with frozen salineanalysis determined that the force necessary to create fracture in asample maintained at about −90° C., at about −80° C., or at about −70°C. could range between about 30 N and 90N, specifically 43-72 N with anintended bit design. This range arises from observed variation infracture toughness of experimentally frozen sample, based on observeddata.

Several design alternatives were investigated, including drills havingmotion that is rotary or linear oscillatory.

Example 2 Rotary Drill

The rotary motion method of coring is accomplished by driving a coringbit with a rotary motion (i.e. drill press). This method requires bothhigh axial and torsional loading of the core bit to penetrate thesample. Attempts at rotary drilling of frozen biological specimensshowed some limited success, generating few successful samples asdetermined by analytical data. Additional data from analysis of thelinear oscillatory motion are shown herein.

Example 3 Linear Oscillatory Motion

A linear oscillatory ultrasonic drilling system provided herein includesa piezoelectric stack, which drives a horn to a resonant frequency.Typical ultrasonic systems operate at frequencies above 20 KHz withdisplacements up to 30 μm. Examples herein of sampling using acommercial ultrasonic polisher found excessive amount of heat, confirmedby computation. The ultrasonic polishing device was equipped with adrilling bit and introduced into the frozen sample. The devicecompletely melted the frozen specimen. The adjustability and controlover the parameters of motion is also limited.

Therefore, another example of a type of linearly oscillating systeminvestigated herein was a digitally controlled linear motor system. Thistype of motor can be used to drive a system at subsonic speeds andprovide the motion profile required. An exemplary motor of this typesuitable for the methods herein is manufactured by the LinMotCorporation (Delevan, Wis. 53115), and provides a tubular linear motorusing a cylindrical stator to drive a hollow slider containing neodymiummagnets. An advantage of this motor is added control over the parametersof motion.

Example 4 Exemplary Apparatus

A linear motor, for example from LinMot Corporation, produces a sonic,linear oscillatory motion for sampling. The stator and slider assemblyis operated by a servo controller which communicates and is programmedthrough the COM port of any PC equipped with the provided software.Analytical investigations were performed to determine optimizedconformance to the objectives.

Initial calculations were used to determine the amount of heat generatedin the system. Possible modes of heat generation in the system include:fracture, friction, and comminution. A negligible amount of heat isgenerated from fracture because of the brittle nature of the material.Heat from friction was calculated to be directly proportional to thefrequency and stroke of the motion. Calculations showed that theultrasonic method of coring produces 27 times more heat from frictionthan the LinMot system. Comminution arises from the mechanical sizereduction of the sample particles, which will occur as the bit coresinto the frozen sample. The heat from comminution is calculated usingthe Rittenger Equation, in which energy methods are used to relateinitial and final chip surface areas to an amount of heat generation. Asthe Rittenger method was developed empirically, it is difficult toquantify heat generated from comminution in this system. In addition toheat generation, heat transfer from chip transport was calculated withthe known boundary conditions of the system, including size of the chipsand velocity of chip flow. As with heat generated from comminution,transfer of heat from chip flow is optimally determined through testing.

The critical force necessary to initiate fracture at −79° C. wasdetermined by the following equation.

Critical force=P=[K _(1C) /A]*[HV/E] ^(1/2) *Co ^(1.5)

This relates the fracture toughness, Vickers hardness, and an assumedresultant radial crack length of the material, to determine the criticalforce to cause fracture.

Due to a range of fracture toughness values for ice, a range of thenecessary force was determined to be between 43 N and 72 N. To supportthis, finite element analysis using ANSYS was computed. The lower limitof 43N from the above critical force calculation was applied to aninfinite sheet of ice and the resulting principle stresses werecomputed. This ANSYS analysis produced compressive stresses of 60 MPa.Empirical observations of ice fracturing under uni-axial compressionshow a necessary stress of 4-30 MPa. This value is less than the 60 MParesulting from the 43 N force. The combination of the critical forcecalculation and ANSYS analysis, indicated suitability of a LinMot drivensystem.

Example 5 Free Fall Force

A free-fall force approach was conducted as an additional means ofdetermining the force necessary to fracture frozen saline. Using thework-energy theorem and conservation of energy, it was calculated thatthe experiment imparted 73 N of force to the frozen saline. Fractureoccurred and ice chips were produced, with an indentation that wasapproximately twice the diameter of the bit. This test provided furtherverification of the theoretical analysis.

It was concluded that an advantage of the LinMot driven design conceptis control over motion, achieved through the manipulation of thesystem's software. The LinMot system is exemplary for oscillatory motionwith minimal heat generation. In addition, the LinMot is equipped with ahollow slider design that allows for removal of the frozen sample afterit is obtained.

Example 6 Improvements and Equivalents

A servo memory for the LinMot or equivalent systems is upgraded to allowfor more data points. The LinMot controller runs a motion profile thatcontains 4000 data points. An increase in data points enables the systemto operate over a larger range of frequency, amplitude, and force whilealso providing a feed rate.

Other beneficial improvements are use of titanium as the bit material toincrease the hardness of the bit by 45%. Additional modifications to thebit geometry allow the bit to remove chips from the sample whileobtaining a sample.

Another improvement is to place a hydrophobic coating on the bit, suchas Teflon. This reduces friction and allows easier sample recovery fromthe bit.

Example 7 Bit Impact Test

Using the LinMot subsonic impact driller, the system was mounted in astationary position such that the slider of the LinMot was the onlymoving part. LinMot software includes force feedback of the amperage anddriving force levels of the systems.

Several single impact analyses were conducted to determine the pressurerequired to fracture the ice surface with minimal radial cracking. Thetest set-up included several operations at various average speedsranging from 1.22 to 240 mm/s. A 30° conical stud needle made of 316stainless steel was threaded into the LinMot slider. To measure theincrease in pressure on the surface of the ice, the tip of the studneedle was ground down to a flat surface to increase the surface area incontact with the ice at impact.

To determine the impact velocity the average velocity of the last threedata points immediately preceding impact with the ice were considered.The average impact velocity was found to be 128 mm/s. The driving forceat the point of contact was determined by using a conversion factor fromthe software and amperage spike in the output. The amperage at thesurface of the ice was found to be 1.47 A. The driving force wasdetermined to be 29.48 N. The mass of the slider was also taken intoconsideration as a contributor to the impact energy through momentum.The mass of the slider is 0.5 kg. The momentum was calculated to be0.064 kg*m/s, using the driving force and mass of the slider. Thecalculated impact time was 0.006 seconds. The impact force and pressureof the system was determined by dividing the momentum by the impact timelength.

The highest inertial forces are generated near the surface of the icewhen the slider is extended to the furthest extent from the stator. Theoptimum ice surface fracture profile was at 120 mm/s. The maximum impactarea was determined through testing to be 2.374 mm² and resulted inimpact force of 40.153 N, a resultant pressure of 10 MPa.

Example 8 Bit Analysis

A factorial design of experiments was performed to determine howcritical factors of the bit geometry affected the stress in the bitduring impact as shown in Table 1 herein. Six independent factors werechosen, A: outer diameter, B: impact angle. C: fillet radius, D: toothheight, E: impact width, and F: tooth angle.

A fractional 2 k factorial design experiment was used. For each factor ahigh-level and low-level dimension was indicated. This resulted in 64combinations that were reduced using the half fraction of the fullfactorial design based on six factors. The 32 combinations were analyzedin ANSYS to solve for maximum Von Mises stress.

TABLE 1 Combination maximum stress results showing the high-lowcombinations and the results. A B C D E F Max Stress (Mpa) 1 −1 −1 −1 −1−1 −1 321.30 2 −1 −1 −1 −1 1 1 106.70 3 −1 −1 −1 1 −1 1 345.60 4 −1 −1−1 1 1 −1 118.47 5 −1 −1 1 −1 −1 1 371.00 6 −1 −1 1 −1 1 −1 123.60 7 −1−1 1 1 −1 −1 451.80 8 −1 −1 1 1 1 1 132.50 9 −1 1 −1 −1 −1 1 692.00 10−1 1 −1 −1 1 −1 209.00 11 −1 1 −1 1 −1 −1 1,731.00 12 −1 1 −1 1 1 1363.82 13 −1 1 1 −1 −1 −1 935.38 14 −1 1 1 −1 1 1 255.52 15 −1 1 1 1 −11 4,034.00 16 −1 1 1 1 1 −1 521.39 17 1 −1 −1 −1 −1 1 321.16 18 1 −1 −1−1 1 −1 116.03 19 1 −1 −1 1 −1 −1 379.33 20 1 −1 −1 1 1 1 115.63 21 1 −11 −1 −1 −1 403.87 22 1 −1 1 −1 1 1 119.31 23 1 −1 1 1 −1 1 415.38 24 1−1 1 1 1 −1 144.62 25 1 1 −1 −1 −1 −1 755.18 26 1 1 −1 −1 1 1 198.71 271 1 −1 1 −1 1 1,641.00 28 1 1 −1 1 1 −1 328.76 29 1 1 1 −1 −1 1 925.2730 1 1 1 −1 1 −1 247.16 31 1 1 1 1 −1 −1 3,478.00 32 1 1 1 1 1 1 542.18

TABLE 2 Combination maximum stress results Outer Impact Fillet ToothImpact Tooth Diameter Angle Radius Height Width Angle Avg of 681.401,128.45 888.66 1,004.70 227.63 714.27 High Average 238.59 202.96 215.35206.61 423.45 240.04 Low Main 442.82 925.49 673.31 798.09 −195.82 474.23Effects

The main effects of these six factors were calculated using the resultsshown in Table 2. For example the main effect of the outer diameter wascalculated by subtracting the average stress of the combinations, wherethat factor is a high value, from the average stress of the combinationswhere that factor is a low value. Table 2 shows the results of thesecalculations, wherein the numerical results are the average stressexpressed in MPa.

The outer diameter and the impact angle were found not to have mucheffect on the stress output. Stress decreases as the impact width isincreased. The remaining factors show an increase in stress as thedimension of the factor is increased. Of these factors, the tooth angleshows the greatest effect. The tooth height has less of an influencethan the tooth angle and more than the fillet radius. These resultsalong with maximum surface area to break ice during impact were used tooptimize the bit geometry.

Example 9 Manufacturability and Material Selection

Drilling bit designs were analyzed and were optimized for ease ofmanufacturability by utilizing available standard tubing sizes,adjusting tolerance levels, and designing the bits with commondimensions.

Bit designs were developed to compare thick wall and thin wall designs.These designs vary in only two or three dimensions. Consistentmeasurements allow for ease of manufacturing by limiting set up time.Variation between the bits is limited to dimensions that affect theperformance of the device. Testing determines the features that arebetter suited to the application.

The bit is made of prefabricated standard surgical tubing. Availablematerials include titanium, INCONEL 625, and each of the stainless steel304, 304L, 316, 316L. Table 3 shows the material properties of fourmaterials at room temperature. The drilling bit utilized in thisapplication will be subject to low temperatures. The 300 series ofaustenitic stainless steels (characterized by high ductility, low yieldstress and high tensile strength) are important because they retain alarge portion of their room temperature toughness at temperatures as lowas −252° C.

TABLE 3 Material properties of stainless steels Modulus of Yield BrinellStainless Steel Elasticity Strength Endurance Limit Hardness Materials[Gpa] [MPa] [MPa] [HB] 304 200 205 259 201 316 200 205 259 217 304L 200170 244 201 316L 200 170 244 217

The material properties in Table 3 show that stainless steel 316 is anoptimal material. Stainless steels 304 and 316 have the largest yieldstrength and endurance limit. The 316 stainless steels are hardermaterials, for the use herein is advantageous. The 316 stainless steelhas optimal values and is widely used in the biomedical industry.Further, the 316 steel is more resistant to general corrosion then the304 stainless steels. Common tube sizes are commercially available froma variety of sources including N.E. Small Tube (Litchfiled, N.H.),MicroGroup (Medway, Mass.), Unimed (Solvay, Brooklyn, N.Y.), and Popperand Sons (New Hyde Park, N.Y.).

The bit design includes a tip geometry that meets several criteria,including maximizing crack propagation axially into the frozen specimenwhile minimizing the propagation in the radial direction; bit life; andoptimizing material characteristics, including diamond coating optionsand other additions as is beneficial to requirements.

Example 10 Bit Design

A main objective is to obtain a sample equivalent of 110 μL (100 mm³)+10of a specimen. Using a standard 60 mm in length cryotube, the diameterrequired for the drilling function of the needle is approximately 1.9mm, calculated as shown herein using the volume of the needle cylinder.The length of frozen material available in a 60 mm cryotube isapproximately 35 mm.

V_(CYL) = π r²L$r = \sqrt{\frac{100\mspace{14mu} {mm}^{3}}{\pi*35\mspace{14mu} {mm}}}$r = .954  mm D = 1.908  mmV_(CYL) = Coring  Volume = 100  mm³r = Inner  Radius  of  Needle  (mm)D = Inner  Diameter  of  Needle  (mm)

A bit used for initial testing included a four inch long needleconnected to an unthreaded brass slug with a flat diamond coated tip.The needle was made from 303 stainless steel tubing with an innerdiameter (ID) of 1.0668 mm and an outer diameter (OD) of 1.9812 mm. Thisdesign was found to have long cyclic loading life of the bit, anduniformity of crack length propagation at impact due to the circularnature of the impact surface. However this flat impact face was found tobe inefficient in producing successful samples with a model system,frozen saline solution.

A further bit design utilized the same ID and OD as herein, with athreaded brass slug pressed onto the needle to facilitate attachment,and a tip ground down to a 75° point. This dual tip design showedimproved performance compared to the design herein in ease of use andlength of the obtained frozen sample of approximately 15.875 mm.

Improvements to the dual tip design included the following: reducingstress in the bit so to achieve longer bit life, improving the cuttingeffectiveness of the system to shorten sampling time, and incorporatingan ice chip removal system to clear unwanted frozen chips from theimpact area to prevent binding of the needle. The embodiment hereinfurther addresses these issues.

Example 11 Exemplary Drilling Bit

An exemplary drilling bit was used that includes a wall with a 2.5 mm ODand a 1.80 mm ID, 90° cuts yielding 4 tips which do not come toinfinitesimal points but rather have flat impact surfaces ofapproximately 0.3 mm thickness. This feature reduces the maximum stressin the bit upon impact. Additionally, 0.25 mm fillets at the cuts werefound further to reduce stress.

Lifting and ejection of the frozen sample model of saline is facilitatedthrough the use of lifting teeth spaced at 1 mm intervals herein theimpact surface along the entire length of the bit. To make contact withthe frozen sample a lifting tooth having a diameter of 2.7 mm was made,which is a 0.1 mm larger diameter than the remaining lifting teeth. Thismodification was made to create a buffer zone below which no cleared icechips may re-enter and cause the binding of the needle. The air gapbetween the cylinder wall of frozen sample and the smaller lifting teethfunction to assist in the removal of ice chips through air vorticescreated by the oscillatory Z-axis motion of the needle. The height ofthe first lifting tooth was changed to 0.1 mm in order to eliminate thelarge deflection seen at the lifting tooth, ice interface in observedstress analyses. The angles between the lifting teeth and the needlecylinder wall are at 22° in order to provide a steep slope for an easiertransition of the ice particles between the lifting teeth and frozensample cylinder wall.

Stress analyses were conducted on the quad tip design to validateselection of the particular geometry. As dual tip design exhibited amaximum von Mises stress of 1978 MPa and a maximum deflection of 0.254millimeters, when the maximum stress required to fracture frozen salinesolution in tri-axial compression of 60 MPa was loaded as a pressureforce onto the contact faces of the bit. The maximum stress experiencedby the needle equated to 179,281 cycles to failure for the bit. Usingthe average time to extract a core of 150 seconds at 60 Hz this equatesto 9000 cycles per core and roughly 20 cores to failure. This analysiswas reproduced with the prototype design to determine the improvementsin cyclic loading.

To accurately simulate an initial impact test, a pressure of 345 MPa(see Table 3 herein) was added as a structural load to all the impactfaces of the contact bit. This load simulates an upper bound for theforces that will act on the needle tip. This pressure was calculated bytaking the maximum force the LinMot can apply to the system, 120Newtons, and applying it to the surface areas of initial contact.

TABLE 3 Impact analysis pressure load calculation P = F/A P = Pressure(Mpa) P = 30 N/.0869 mm² F = Force (Newtons) P = 345 N/mm² (Mpa) A =Area (mm²)

The bit model was constrained at the top in the X, Y, and Z axes as wellas rotations in all the axes to accurately simulate the physicalconstraints that are applied to the needle by the LinMot system.

This design generates a maximum von Mises stress of 354 MPa, a reductionof 82% over the previous dual tip design. The largest deflection in themodel is 0.0026 mm, a reduction of 99% over the previous needle design.As is evident, the maximum stress occurs above the impact faces of thebit.

Example 12 Fatigue Life

The maximum Von Mises stress seen in this bit was used to determine alower bound for the number of cycles to failure, N_(f), of the needle.The maximum stress for the previous needle was equivalent to 179,281cycles to failure for the bit. Using the average time to obtain a sampleof 150 seconds at 60 Hz this equates to 9000 cycles per sample and 19.92cores to failure. Analysis yielded a maximum stress of 354 MPa. The bitexperiences stress solely in compression and not in tension, thereforethe alternating stress is half of the maximum stress. Utilizing thealternating bit stress the Basquin Equation was employed to determinethe cycles to failure of the bit.

Test results show that the N_(f) of the bit yields 2,003,197 cycles tofailure of the bit. At 7500 cycles per sample for the design (50 Hz at150 seconds per sample) this is equivalent to 267 samples before failureof the bit occurs, an improvement of 1335% over the previous design.

Example 13 Automation

An embodiment of the method and apparatus herein includes controllingZ-axis motion through an actuator and MCS. A load cell is incorporatedto gather force measurements near the point of impact. The controlsystem include an MCS capable of operating both actuators and processingthe output from the load cell.

Force data during the drilling operation provides information about thefriction force along the core shaft. Data gathered allows adjusting thebit geometry based on maximum impact force measurements. The load cellmust withstand vibrations of 50 Hz as this is the operating frequency ofthe LinMot actuator. It must also take force measurements at a rate ofspeed greater than 200 Hz in order to provide sufficient informationabout the impact event. The maximum force that can be generated by thesystem is 120 N of force, combined with the momentum energy of theslider and the coring bit. Minimal deformation under loading will assistin isolating impact deflection from deflection of the load cell.

Actuation of the LinMot is controlled primarily through the embeddedcontroller in the amplifier. This controller is calibrated to calculateoutput currents based on the mass of the slider and the input motionprofile. The added mass of the load cell and the drilling bit is notaccounted for when the controller calculates a move. Therefore, everymove will now have an amount of reactionary motion due to the extrainertia the controller is not expecting. The oscillating mass increasesby approximately 113.

Deflection of the load cell is minimized to enable measurement of smallscale changes in the waveform. The load cell should not deflect duringloading to maximize the energy transmitted to the frozen sample.Preliminary investigation of impact characteristics indicates thatimpact depth, the distance into the ice the bit travels each impact, isabout 0.04 mm. This translates to a gross motion velocity of 2 mm/s. Thelow distance helps to minimize radial ice fracture by reducing crackpropagation length in all directions.

The apparatus is programmed to oscillate a 2 mm amplitude sine wave at afrequency of 50 Hz. The frequency response of the load cell is higherthan the frequency of the drilling operation to allow for propermeasurements. To characterize impact dynamics, force data is gathered atleast 4 times a cycle. The sample frequency is greater than 200 Hz. Theacquisition instrument must also be considered at this point in time.

During initial testing, LabView is utilized to process the data from theload cell in order to allow the team to characterize coring operations.However any load cell suitable for use with LabView is suitable for usewith other control systems. Many load cell manufacturers offer deviceswithin the design specifications for example a piezoelectric load cellfrom PCB Piezotronics (Depew, N.Y.) for this application due to theshort lead-time and the qualifications of the quartz force sensor.Piezoelectric load cells are based on the electromechanical response ofcertain crystals to dynamic loading. Force measurements are made byobserving the variation of supply voltage to a crystal while it isdynamically loaded. The crystal, quartz in this case, exhibits varyingconductivity, which is measured as a change in the supply voltage at thepower supply. This variation is then converted into an analog DC voltageoutput. Commercially available load cells such as the PCB are availablein a range of types and the power supplies to go with them. Systemintegration, on the electric side, involves plugging the amplifieroutput into LabView. The apparatus then has capability to monitor forceduring impact and adjust the coring profiles appropriately. The PCB221B02 quartz force sensor is suitable for the design requirementsherein.

The linear actuator is rigidly connected to the supporting structure,oriented vertically. The exemplary LinMot is rigidly connected to themounting plate. The application of force is approximately 50 mm distancefrom the front surface of the mounts. An approximation of the maximumforces possible during coring yields an axial force of 300 N and amoment of 15 Nm across the carriage. There is negligible moment aroundthe axis and negligible force in the x- or y-axis.

A fully integrated automated system embodiment having a force feedbacksensor is tested. The LinMot subsonic impact driller is set at afrequency of 50 Hz. The incorporation of a force sensor provides usefulfeedback on areas of high stress within the ice and assist indetermining the optimal motion profile.

Quantification of a fully automated system versus a manually operatedsystem involves trial tests of the needle bit having the designdescribed herein. Using the LinMot impact driller at the determined 50Hz profile, and as a variable in the testing procedure, a break ofbetween the 0.05 mm steps controlled by the Z-axis was performed, asshown in Table 5 herein.

TABLE 5 Motion profile testing Step Size Break Frequency Drill Depth[mm] [s] [Hz] [mm] Results 0.05 0.2 50 16.7 bound up 0.05 0.3 50 14.8bound up 0.05 0.4 50 21.4 bound up 0.05 0.5 50 25.3 successful

A change in the break of the Z-axis motion controller concluded in asuccessful core to a depth of 25.3 mm. Another test increased the stepof the Z-axis to increase the rate of drilling and constrain thefrequency of the LinMot to 50 Hz and the break of the Z-axis to 0.5seconds, with results shown in Table 6 herein.

TABLE 6 Time efficiency increases in drilling Step Size [mm] Break [s]Frequency [Hz] Drill Depth [mm] Results 0.1 0.5 50 25.0 successful 0.150.5 50 25.0 successful 0.2 0.5 50 24.3 successful 0.25 0.5 50 24.9successful 0.3 0.5 50 25.3 successful 0.35 0.5 50 25.0 successful 0.40.5 50 24.7 successfulThe conclusion of these tests and time efficiency provided an optimalmotion profile.

The automated system of the needle drilling is an improved tool foraliquotting of frozen biological specimens. The drilling bit geometrywas optimized through virtual design and by experiment testing. Theresults were factored to yield the main effects of each feature withrelation to maximum stress in the coring bit. Results showed that impactwidth and tooth angle were most important in effecting the stress in thebit during impact. These results drove optimizing bit for fatigue lifeby reducing stress while maintaining required impact area to break ice.The optimized prototype bit exceeds the satisfactory usable lifespan of150 samples

Example 14 Tissue Microarrays (TMA) from Frozen Specimens

TMAs (tissue microarrays) have been used throughout the world forseveral years. The benefit of placing hundreds of tissue core samples inone block now has proven time and again to be invaluable in the researchof large study cohorts. The H&E (Hematoxylin and Eosin) stained slideprepared on one TMA block has been used to reveal diagnostic informationfor hundreds of patient cases. Additionally the TMA is gainingpopularity for use in areas beyond clinical research. For example, inanalysis of genes, antibodies and proteins in a large cohort of, forexample, 3,000 patients, the TMA is a method of reviewing multipletissue types and diagnostic features from the saline preparation.Conversely, staining 3,000 individual slides to review only oneparticular antibody is not only antiquated, but prohibitive in terms oflabor cost in technical time and for reagents. The TMA has earned asignificant place in the research environment and is useful in theclinical field as this method fulfills the need for high throughput andaccuracy in the clinical laboratory grows.

While the present methods pf TMA involve paraffin embedded tissue, thereare drawbacks to handling fixed tissue in paraffin. While mosttechniques in the past were designed for fixed tissue now we are seeingan increased need for frozen tissue to be used in the place of fixedmaterial. In order for a tissue to be embedded in paraffin, it has to gothrough a process of fixation and processing as well as clearing andinfiltration at high temperatures in liquid paraffin. These processeshave the effects of decreasing the antigenicity of the tissue as well ascross linking the proteins and degrading other tissue structures, withthe negative outcome that many studies can not be done on the fixedspecimen. Pathology analysis has long become attuned to the architectureof the formalin fixed specimen and problems that lie therein. However,there is a need for fresh frozen tissue microarrays (ffTMAs) in order toperform studies for many of the more labile antigens, proteins, DNA,RNA, mRNA, peptides to name just a few.

The optimal specimen in the pathology laboratory is the snap frozenfresh tissue specimen, which is stored in the −80° C. freezer. Thisspecimen may be used for any investigational process at any time. Thefrozen sample remains in a state of biological functionality and isready for investigational use, after days, months, or years fromfreezing. Frozen sections may be cut with the use of a cryostat toobtain a diagnostic section to evaluate the entire surface of thespecimen, or for special studies for DNA and RNA extraction, or for FISHand ISH immunofluorescent techniques. Individually sectioned slides maybe fixed for specific procedures resulting in optimal test results andless time removing undesired effects of improper fixation. Smalleramounts of this precious tissue are required for sectioning therebysaving the remaining specimen for future work. Ultimately, a smallportion of the fresh frozen specimen may be fixed and processed for apermanent pathological record, thus, allowing for the fixed tissue to beused in paraffin TMA in years to come.

The versatility and breadth of techniques available makes the freshfrozen specimen the most valuable to the research and diagnosticlaboratory. Most general pathology laboratories and especially those inacademic centers snap freeze a portion of fresh tissue samples whenpossible. These tissues are of special interest due to the specificdiagnosis. Examples of such specimens are lymph nodes, prostate tumors,breast tumors, nerve and muscle.

There are large repositories of tumors and fresh frozen specimenslocated in academic centers for both diagnostic and research purposes.However, there is a need for consistency of specimen handling in orderto maintain diagnostic integrity of the tissue for valid interpretativeresults.

Cell line arrays have been constructed herein and frozen cell linearrays were experimentally shown to be suitable for determining proteinphosphorylation in frozen samples. Protein Phosphorylation is animportant and useful mechanism to demonstrate however, the results arevery unreliable in formalin fixed samples.

A multi tumor array was constructed that included samples of tissuesfrom sources such as prostate cancer benign prostate, colon, renalcarcinoma, liver, and lung. The process of constructing the arrayyielded a ffTMA frozen tissue microarray for which the results of thefinal product yielded beneficial data, for example, a test for Hepsinimaging peptides which would have been otherwise impossible to obtainwith fixed tissue. Protein antigens are often destroyed during fixationor require strong antigen retrieval.

Referring to the construction of the frozen multi tumor array, arrayswere created herein using preserved in the −80 freezer tissues. Resultsare equivalent between fresh frozen tissue and those frozen more thantwo years previously showing the most valuable points of frozen tissue.

The multi tumor array was constructed using the manual Beecher MTA1arrayer and OCT compound (Optimal Cutting Temperature, from TissueTek),a water soluble embedding medium, used as the embedding medium and isthe medium used for maintaining the specimens in the frozen state, whichhelps to avoid desiccation in the −80 freezer. In order to keep the areaaround the arrayer cold enough to maintain a temperature suitable to theintegrity of the tissue, dry ice was used in and around the arrayer. Thetissue samples as well as the array were monitored for temperaturefluctuations as to ensure temperature control. The array was preparedsimilarly to a paraffin array, in that a mapping and file weremaintained of all specimens and locations. Samples were removed from thedonor frozen specimen blocks and were placed in to a recipient embeddingwell. The array enabled experiments with this frozen multi tumor arraythat would be impossible by any prior art technology, treating an entireselection of tissues at one time on one slide with Hepsin imagingpeptides. By prior techniques, many sections of individual tissues wouldhave been required to analyze the value of various tissue types and thepotential for example controls.

The frozen samples are sectioned on a research quality cryostat (LeicaMicrosystems, Heidelberg, Germany). After each section is cut it is thenpicked up with the use of the CryoJane Tape Transfer System(Instrumedics Inc., Hackensack, N.J.). The tape transfer system allowsfor the secure adherence of the tissue to the slide, which is alsoprovided by Instrumedics. There is a greater yield of samples per slideand much less waste of tissue samples using the CryoJane system andslides.

Frozen Cell Line Arrays

Protein phosphorylation is an important mechanism for cellularsignaling. Current methods for determining phosphorylated proteins oftenyield unreliable results. Intracellular phosphatases andformalin-fixation dephosphorylate these proteins within milliseconds ofischemia which occurs when cells or tissue are harvested. The additionof phosphatase inhibitors does not seem to not stop this process. Onemethod to preserve the phosphorylation status is to harvest tissue orcell lines, add phosphatase inhibitors and immediately decrease thetemperature by placing the sample on ice and freezing it.

To generate the frozen cell line arrays 30 μl of a cell line suspensionin phosphate-buffered saline is transferred with a 20 μl pipette tipinto the wells of the mold (panel A in FIG. 4). Currently, we are ableto array up to 16 separate cell line samples at once. Panel B in FIG. 4depicts a core of this array with an H&E staining. Since cell linesconsist of one single cell clone obtaining a solid tissue core is notnecessary. In panel C of FIG. 4, a high magnification of a cell linecore stained with an antibody specific for the phosphorylated form ofthe extracellular-regulated kinase (ERK) is shown. Strong nuclearstaining can be appreciated.

Multi-Tumor Frozen Tissue Array

Monitoring expression of Hepsin, that is a biomarker over-expressed inprostate cancer, would be extremely useful in the diagnosis of prostatecancer. Although PSA is being currently used in standard screening testsfor prostate cancer, it is not always a good indicator. Hence there is aneed for developing a non-invasive method for the diagnosis of prostatecancers. Hence Hepsin-specific peptides have been identified for in vivoimaging.

Peptides binding to Hepsin were identified by phage display technology.The binding of these peptides to Hepsin was confirmed using cell linesthat over-express Hepsin. However, in order to develop an imaging agent,it is important to demonstrate that the peptides are capable of bindingto the protein antigen in human tissues. The major obstacle to testingthe peptides on patient tissue samples or tissue microarrays (TMA) isthat these peptides are confirmation specific and require an intactantigen for optimal binding. The TMAs constructed using formalin fixedparaffin embedded (FFPE) tissues cannot be used for testing suchpeptides since protein antigens are often destroyed during the processof fixation and antigen retrieval. Although most antigens retain aconformation that can be recognized by large antibodies for routineimmuno histochemistry (IHC), the antigens that are peptides are smallermolecules, and require an intact local conformation. Hence such studiesrequire frozen material as local conformations are well preserved infrozen tissues.

In order to test candidate Hepsin binding peptides for in vivo imaging,a frozen multi-tumor TMA consisting of tumor material from prostate,lung, colon, liver, renal, breast and benign prostate tissue constructedmanually at our lab was used. FIG. 5 shows specific staining of Hepsinusing the FITC-labeled peptides in cancer glands. Specific membranousstaining of Hepsin can be seen in the luminal side of the cancer glandswhich is markedly absent in the benign gland.

Hence, data herein show successful test potential for Hepsin imagingpeptides using a frozen TMA. The multi-tumor frozen array constructedfor this study also offers a potential to test markers in diversetissues for imaging of different types of cancers.

1-26. (canceled)
 27. A frozen tissue micro-arrayer comprising: a coringbit; a robotic positioning system configured to move the coring bit intoa frozen tissue specimen while the tissue specimen remains frozen to cuta frozen sample core from the frozen tissue specimen, withdraw thecoring bit from the frozen tissue specimen while the frozen sample coreis retained in the coring bit, and move the coring bit and the frozensample core to a destination location for receiving the frozen samplecore; and an ejection system configured to eject the frozen sample corefrom the coring bit to deposit the frozen sample core at the destinationlocation, wherein the robotic positioning system is adapted to controlforces associated with the cutting of the frozen sample core from thefrozen tissue specimen to substantially maintain an initial tissuemorphology of the frozen tissue specimen during the cutting process. 28.A frozen tissue micro-arrayer as set forth in claim 27 wherein theejection system is adapted to control forces associated with ejection ofthe frozen sample core to substantially maintain the initial tissuemorphology during the ejection process.
 29. A frozen tissuemicro-arrayer as set forth in claim 28 wherein the forces associatedwith cutting and ejection are controlled so there is no detectablealteration of the initial tissue morphology in the frozen sample corewhen it is ejected from the coring bit.
 30. A frozen tissuemicro-arrayer as set forth in claim 28 wherein the ejection system isadapted to drive the frozen sample core into a recipient block as it isejected from the coring bit.
 31. A frozen tissue micro-arrayer as setforth in claim 27 further comprising a system for pre-cooling the coringbit before the robotic positioning system begins moving the coring bitinto the frozen sample.
 32. A frozen tissue micro-arrayer as set forthin claim 27 wherein the micro-arrayer is configured to maintaintemperature of the frozen sample core at a temperature no warmer than−20° C. from the time the coring bit is moved into the frozen tissuespecimen until the frozen sample core is ejected at the destinationlocation.
 33. A frozen tissue micro-arrayer as set forth in claim 32further comprising a cooling system adapted for receiving the frozentissue specimen.
 34. A frozen tissue micro-arrayer as set forth in claim32 further comprising a system for pre-cooling the coring bit before therobotic positioning system begins moving the coring bit into the frozentissue specimen.
 35. A frozen tissue micro-arrayer as set forth in claim27 wherein the robotic positioning comprises a motor configured to drivea rotary motion of the coring bit as it moves the coring bit into thefrozen tissue specimen.
 36. A frozen tissue micro-arrayer as set forthin claim 27 wherein the robotic positioning system comprises a motorconfigured to drive a linear oscillating movement of the coring bit asthe robotic positioning system moves the coring bit into the frozentissue specimen.
 37. A method of making a frozen tissue micro-array, themethod comprising: moving a coring bit into a frozen tissue specimenwhile using a motor to drive a cutting action of the coring bit so thata frozen sample core from the frozen tissue specimen is cut from thefrozen tissue specimen and received in the coring bit; withdrawing thecoring bit from the frozen tissue specimen while the frozen sample coreis retained in the coring bit; moving the coring bit and the frozensample core retained therein to a destination location; and ejecting thefrozen sample core from the coring bit and depositing the frozen samplecore at the destination location, wherein forces associated with cuttingof the frozen sample core from the frozen tissue specimen are controlledto substantially maintain an initial tissue morphology of the frozentissue specimen during the cutting process so that the frozen samplecore has a morphology representative of the morphology of the frozentissue specimen at the location where the coring bit moved into thefrozen tissue specimen.
 38. A method as recited in claim 37 furthercomprising using the motor to drive a rotary cutting motion of thecoring bit as it is moved into the frozen tissue specimen.
 39. A methodas set forth in claim 37 further comprising using the motor to drive alinear oscillating cutting movement of the coring bit as it is movedinto the frozen tissue specimen.
 40. A method as set forth in claim 37wherein the destination location is a recipient block of a micro-arrayand the ejecting and depositing comprises depositing the frozen samplecore in the recipient block.
 41. A method as set forth in claim 40further comprising controlling forces associated with the ejecting anddepositing the frozen sample in the recipient block to substantiallymaintain an initial tissue morphology of the frozen tissue specimenduring the depositing so that the deposited frozen sample core has amorphology representative of the morphology of the frozen tissuespecimen at the location where the coring bit moved into the frozentissue specimen.
 42. A method as set forth in claim 37 furthercomprising pre-cooling the coring bit before moving the coring bit intothe frozen sample.
 43. A method as set forth in claim 37 furthercomprising maintaining a temperature of the frozen sample core at atemperature no warmer than −20° C. from the time the coring bit is movedinto the frozen tissue sample until the frozen sample core is ejected atthe destination location.
 44. A method as set forth in claim 37 furthercomprising using a robotic positioning system to move the coring bitinto a frozen tissue specimen, withdraw the coring bit from the frozentissue specimen, and move the coring bit and the frozen sample coreretained therein to the destination location.
 45. A method as set forthin claim 37 further comprising measuring applied force and torque duringas the frozen sample core is cut from the frozen tissue specimen.
 46. Amethod as set forth in claim 37 wherein the frozen tissue specimen is afrozen fresh tissue specimen.